Recombinant human IgM-antibody effective against cancer cells

ABSTRACT

A recombinant human monoclonal pentameric IgM antibody comprising the capability of oligospecific binding to purified ganglioside epitopes GD3, GM3, GD2 and GM1 and the capability of specific binding to malignant cancer cells selected from the group consisting of melanoma cells, small cell lung cancer cells, glioblastoma cells, and estrogen receptor-negative metastatic breast cancer cells; a cell line producing the IgM antibody; and the use of the IgM antibody as a diagnostic tool and/or as a therapeutic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. §§ 371 national phase conversionof PCT/EP2013/074762, filed Nov. 26, 2013, which claims priority to U.S.Provisional Patent Application No. 61/731,586, filed Nov. 30, 2013, thecontents of both of which are incorporated herein by reference. The PCTInternational Application was published in the English language.

TECHNICAL FIELD

The present invention relates to the manufacture and use of IgMantibodies having a binding specificity for a number of gangliosides andbeing effective against various malignant tumor cells and cancers.

STATE OF THE ART

Gangliosides are complex glycolipid constituents of cell membranes. Theyare involved in many biological functions including cell-cellrecognition, cell-matrix attachment, cell growth and celldifferentiation. They are synthesized in the Golgi apparatus of the cellas a consequence of multienzyme pathways leading to differentsubstructures. Studies of ganglioside expression in different “malignantcells” such as small cell lung cancer cells, melanomas, neuroblastomas,certain breast cancer cells and others reveal deviations of gangliosideexpression as compared to “normal tissues”. Higher expression levels ofgangliosides such as GD3, GD2, GM3, GM1 have been identified in variousmalignant tumors. Apparently, certain mutations in the multienzymepathways of ganglioside expression are contributing to physiologicallyrelevant phenomena determining whether cells or tissues behave as“normal” or as “malignant” phenotypes. Even minor deviations inganglioside expression may cause malignant transformations.

Peter Vollmers and Stephanie Brändlein (Natural antibodies and cancer,New Biotechnology Vol. 25, No. 5, June 2009—Review, Natural IgMantibodies: The orphaned molecules in immune surveillance, AdvancedDelivery Reviews; Vol. 58 (2006)) report that most tumor-specificantibodies which they found belonged nearly exclusively to the IgMclass. The authors also conclude that tumor immunity seems to berestricted to innate immune mechanisms used by nature, like naturalantibodies, which are also most likely considered as excellenttherapeutics. Despite major scientific progress in recent years IgMantibodies still remain somewhat neglected and biased by the establishedantibody researchers community.

European Patent No. 0480440 discloses and claims a monoclonal antibodyagainst melanoma, the antibody comprising an IgM antibody. The presentinvention may thus be regarded as a useful further development of theinvention reported in EP 0480440.

BRIEF DESCRIPTION OF THE INVENTION

The present invention laid down in the independent claims provides for arecombinant human IgM monoclonal antibody that is capable of recognizingdifferent purified ganglioside antigens and that is also capable ofspecifically binding and killing different malignant tumour or cancercells via activation of complement effector function while at the sametime leaving non-malignant (“normal”) cells unaffected.

In one embodiment of the invention the IgM antibody is expressed in itscomplete pentameric relative IgM isoform by a suitable host celltransfected with genetic material obtained from a healthy humanindividual.

In accordance with the present invention, malignant tumour cells such asestrogen receptor-negative breast cancer cells, small cell lung cancercells, melanomas and glioblastomas have been identified as targets forcancer therapy using the present IgM antibodies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation of a viability comparison betweensubclones 1G6 and 2E4 of an anti-GD3-effective IgM-producing CHO cellline after adaptation to serum-free cell culture medium. FIG. 1 Arelates to subclone 1G6, FIG. 1B relates to subclone 2E4; abscissa=timeperiod of cultivation in days; left ordinate and diamond symbols=cellconcentration; right ordinate and square symbols=viability in %.

FIG. 2 is a graphical representation of a comparison in total productyield between subclones 1G6 and 2E4 of an anti-GD3-effectiveIgM-producing CHO cell line after adaptation to serum-free cell culturemedium. FIG. 2 A relates to subclone 1G6, FIG. 2B relates to subclone2E4; abscissa=time period of cultivation in days; left ordinate=sugarconcentration in mg/ml: diamonds=glucose, squares=lactose; rightordinate and triangles=product (antibody) concentration in mg/ml.

FIG. 3 is a graphical representation of a comparison in specificexpression rates between subclones 1G6 and 2E4 of an anti-GD3-effectiveIgM producing CHO cell line after adaptation to serum-free cell culturemedium. FIG. 3 A relates to subclone 1G6, FIG. 3B relates to subclone2E4; abscissa=time period of cultivation in days; left ordinate=specificsugar consumption in nanograms per cell per day (ng/cell/d):diamonds=glucose, squares=lactose; right ordinate and triangles=specificantibody expression rate in pg/cell/d.

FIG. 4 discloses the results of a Generic Assays immuno-dot-blot test ofa cell culture supernatant at different dilutions obtained aftercultivation of the present IgM producing CHO cell line in amini-fermentor (Sixfors, working volume 500 ml). Rows 01 through 03relate to the positive control sample applied at volumes of 10, 50 and100 uL (uL=microliter); rows 15 through 20 relate to supernatant samplesof two different batches, tested at dilutions of 1:6, 1:21 and 1:101,respectively.

FIG. 5 discloses the results of a Generic Assays immuno-dot-blot test ofpurified, i.e. almost 100% pentameric, anti-GD3 IgM antibody at aconcentration of approx. 50 ug/ml). Left lane=purified anti-GD3 IgMantibody (=IgM/GD3); right lane=culture supernatant comprising IgM/GD3at a concentration of approx. 50 ug/ml (ug=microgram).

FIG. 6 is a graphical representation of the experimental resultsobtained from in vitro activation of complement by the present IgM/GD3antibody in the presence of 20% of natural, i.e. non-heat-inactivated,human serum (NHS) as the source of compliment. Ordinate=cell survival in%, 100% being the viable cell count in cell culture medium supplementedwith heat inactivated NHS (hiNHS); abscissa=hiNHS and NHS controlswithout antibody, and various concentrations in the range of from 1 to100 ug/ml of anti-GD3 IgM antibody added to the cell culture of CRL-1690cells.

FIG. 7 is a graphical representation of experimental results analogousto the ones set out in FIG. 6 and obtained under analogous experimentalconditions, however exemplified with COR-L88 cells.

FIG. 8 is a graphical representation of experimental results analogousto the ones set out in FIGS. 6 and 7 obtained under analogousexperimental conditions, however exemplified with HTB-119 cells.

DESCRIPTION OF THE INVENTION

Prior to developing the present invention the cell line disclosed inEP0480440, i.e. deposit no. ECACC 90090703, was obtained and routinelypropagated using ex-vivo medium plus 10% FCS, first in tissueculture-bottles and thereafter followed by spinner culture, forcomparison purposes. However, the IgM-titer achieved with thestate-of-the-art hybridoma was rather low, i.e. in the order of 2-3ng/ml, although the cell growth was within the typical range for thiskind of hetero-hybridomas. In this context it may be mentioned thatinstability of expression in hetero-hybridomas is an often observedphenomenon and may perhaps also count for the low titers in thiscomparative cell culture.

Such low expression titers of antibody production are of course notsuitable for commercial scale manufacture, neither for diagnostic norfor therapeutic application purposes. It was therefore an object of thepresent invention to establish a system for recombinant expression ofthe desired IgM antibody suitable for industrial scale manufacture inorder to provide sufficient amounts of IgM as a tool for cancerdiagnostics and/or for use as a cancer therapeutic agent.

Comparative Example

EP 0480440 discloses that the IgM antibody secreted by the hybridomasreferred to therein is capable of binding to the gangliosides GM3 andGD3. In an attempt to verify said binding specificities a qualified andcommercially available anti-ganglioside dot assay was purchased fromGeneric Assays GmbH, Germany, and was used according to operationprocedures described therein. A 10-fold concentrated culture supernatantwas used.

A slightly positive signal at the GD3 band was indeed identified.Surprisingly, a signal at the GM3 band position was missing contrary toexpections from the disclosure of EP 0480440. A possible explanationtherefore could be that the IgM-titer in the concentrated culturesupernatant (approx. 20-40 ng/ml) was too low for detectable binding toGM3.

Example 1

Given that GD3-overexpression is repeatedly described in scientificpublications for malignant cells such as melanomas it was decided tofocus on the recombinant expression of the antibody of interest. Thegene construct used for this purpose was preliminarily namedPolrecCHOIgM/GD3. The heavy and light chain cDNAs of the hybridomaproducing an IgM binding to GD3 have then been isolated usingestablished techniques of gene isolation and transfection to CHO hostcell lines. The heavy-chain and light-chain polypeptide sequences of thevariable regions are listed hereinafter. Codon optimization forexpression in different host cell species enabled by various state ofthe art techniques has been carried out for various embodiments of theinvention but is not specifically disclosed herein.

SEQ ID NO: 1: PolrecCHOIgM/GD3 heavy chain variable region of mature proteinQVQLVQSGAEMKKPGASVKVSCKASGYTFSSFAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRLTITRDTSASTAYMDLSSLRSEDTAVYYCARNLNYYDILTGLDAFDIWGQGTMVTVSSG SEQ ID NO: 2: PolrecCHOIgM/GD3 light chain variable region of mature proteinDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNKKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQYYST PLTFGPGTKVDIK

The polypeptide sequences of the constant regions of heavy and lightchains are reactive with polyclonal antisera against mu and kappa chainsrespectively.

In order to establish recombinant CHO cell clones expressing theanti-GD3 IgM antibody a rather complex cloning and selection program wasapplied comprising a series of state-of-the-art procedures that havebeen optimized to a certain extent and standardized according to thepresent needs. The cloning and selection program comprised the followingprocedural steps:

-   -   (i) Identification, isolation and sequencing of the genes        encoding the IgM target molecule from the original hybridoma        referred to in EP 0480440, i.e. identified by their deposit        numbers ECACC 90090701 or ECACC 90090703;    -   (ii) Codon-optimization for expression in the host cell-line, in        the present case a CHO dhfr⁻ cell line (dhfr⁻=dihyrofolate        reductase deficient);    -   (iii) Construction of vectors, transfection and selection of        positive CHO-clones;    -   (iv) Several rounds of subcloning and genedose amplification via        MTX-resistance selection;    -   (v) Cultivation of the most promising subclones with respect to        proof of stability of IgM expression and proliferation;    -   (vi) Adaptation and selection of subclones in suitable        protein-free media with continued monitoring of both stability        of IgM-expression and acceptable proliferation activity;    -   (vii) Adaptation and selection of subclones that firmly and        stably express IgM and subsequent propagation of said subclones        in small-scale stirred tank bioreactor environments as suitable        and required for scale-up for industrial manufacture.

This research program has been applied for establishing and selecting arecombinant CHO-clone having the potential for industrial scalemanufacture at a proper quality of a human recombinant IgM antibodyhaving GD3 binding specificity, hereinafter named “IgM/GD3”. In figuresFIG. 1 through FIG. 3 some important parameters of two different IgM/GD3expressing subclones are compared as determined in experiments performedin small-scale stirred tank reactors (Sixfors) operated at a workingvolume of 500 ml.

As can be taken from FIG. 1A and FIG. 1B the subclone “1G6” in thestirred tank mini-reactor exhibited a superior robustness with respectto proliferation in the protein-free medium (batch cultures werecompared) while subclone “2E4” seemed to be rather sensitive in thatphysico/chemical environment, resulting in a sharp decrease in viabilityafter 16 days of cultivation whereas subclone 1 G6 maintained approx.80% viability for 5 more days, i.e for 21 days.

Also, grown under identical culturing conditions the IgM-titers ofsubclone 1 G6 (see FIG. 2A and FIG. 2B) were significantly higher (i.e.325 mg/I after 22 days of culturing) than the IgM-titers yielded withsubclone 2E4 (i.e. 200 mg/I after 18 days of culturing), although thespecific IgM expression rate qP of subclone 2E4 was significantlyhigher, i.e. prevailingly at or above approximately 60 pg/cell/d,relative to the specific product expression rate qP of subclone 1G6,which remained typically in a range of about 30 pg/cell/d (see FIG. 3Aand FIG. 3B).

Although there is certainly quite some potential for furtheroptimization it is evident from the experimental results that therecombinant expression of the IgM/GD3 antibody in CHO host cells isseveral orders of magnitude more efficient than IgM/GD3 production usingone of the original, i.e. state-of-the-art, hybridoma cell linesreferred to in EP 0480440. The recombinant expression procedureaccording to the present invention thus allows for industrial scaleproduction of the IgM/GD3 antibody for both diagnostic and therapeuticapplications.

IgM antibodies are representing extremely large and complex glycoproteinstructures which in the native situation in vivo are correctly expressedand secreted as pentameric isoforms only by certain peripheralB-lymphocytic cells. According to the present inventors' experience itis not obviously derivable nor predictable from prior art knowledgewhether a genetically engineered host cell will be able to recombinantlyexpress complete pentameric IgM-isoforms. Most often times variousisoforms are secreted from a recombinant host cell resulting in amixture of hexamer, pentamer structures and a series of lower molecularweight IgM-fragments. The underlying factors or conditions causingposttranslational fragmentation or incomplete synthesis are stillunknown so far (see e.g. S. Wolbank et al., J. of Virology, Vol.77(2003), p. 4095-4103).

It is therefore stressed in this context that present subclone 1 G6 issecreting the IgM/GD3 antibody as a nearly 100% pentameric isoform. Onlytraces of fragments in the 60 kilodalton range are detected by IgM-SDSPAGE (Novex System; data not shown herein) which traces are also foundin the IgM reference sample derived from human serum.

Also, while the IgM reference sample derived from human serum containeda significant amount of hexameric IgM isoforms such hexameric isoformswere not detected in the recombinant IgM/GD3 fraction of themanufacturing process of the present invention. It can therefore beconcluded that the present recombinant CHO host cell expresses the humanIgM almost quantitatively as a pentamer. In the light of prior artknowledge this is a rather rare and surprising finding (see, forexample, Karola Vorauer-Uhl et al.: “IgM characterization directlyperformed in crude culture supernatants by a new simple electrophoreticmethod”. Journal of Immunobiological Methods 359 (2010, 21-27))

Example 2

In order to confirm the binding specificity of the present humanrecombinant IgM/GD3 antibody the GA Generics Assay Test-Kit forimmuno-dot-blot testing was applied again. The test results are shown inFIG. 4.

Anti-ganglioside dot-blot-test results of recombinant “IgM/GD3”: Culturesupernatants from the 500 ml mini-fermenter (Sixfors) obtained withserum-free nutrient medium have been tested in various dilutions (1:6;1:21; 1:101; see FIG. 4). In all dilutions of the culture supernatant aclearly positive binding to GD3 is visible on the test strip. Later,when purified recombinant IgM/GD3 was available the immuno-dot-blot testwas repeated with purified, i.e. 100% pentameric IgM/GD3 antibody. Ascan be seen in FIG. 5, in addition to its binding capacity with GD3 (seeFIG. 5, right lane) purified IgM/GD3 antibody also yielded a clearlypositive reaction with GM3 (see FIG. 5, left lane). Moreover, there isalso visible some weak binding with GT1a, GD2 and GQ 1b (the latterbetter visible in the original test read-out). Surprisingly, theseadditional binding characteristics are not seen under the experimentalconditions represented in FIG. 4.

There is no scientific basis for explaining the slightly differentbinding results obtained with the crude culture supernatant and itsdilutions (FIG. 4) on one hand and with purified IgM/GD3 antibody (FIG.5) on the other hand. It was therefore assumed that perhaps variationsin quality of the Generic Assays ganglioside immuno-dot-blot test kitscould have accounted for these differing test results. Accordingly, onlypurified gangliosides, as far as available, were used for furthertesting in order to clarify the binding specificities of recombinantIgM/GD3 antibody, which apparently could also be named IgM/GD3/GM3antibody due to its additional GM3 binding specificity. In any case,antibody IgM/GD3 turned out to be also cross-reactive with additionalganglioside antigens or epitopes, respectively, at least to some extent.

The clarification of cross-reactivities is of particular interest sinceaccording to scientific literature natural IgMs, in contrast toantibodies generated by humoral immune response upon infection orvaccination, are generally characterized by limited specificity andrather low binding affinity to their respective epitopes as compared tothe binding affinities of maturated IgGs. Natural IgMs generally showcross-reactivities in binding to different subtypes of antigens orepitopes within chemically related but not identical antigen structures.It is hypothesized that a certain cross-reactivity with generally lowerbinding affinity is an inherent function enabling their role in thegeneral surveillance of the immune response.

Example 3

A series of additional binding tests of the recombinant IgM/GD3monoclonal antibody with purified ganglioside antigens available on themarket have been performed in immunochemical ELISA-test formats. Allpurified ganglioside antigens have been purchased from Calbiochem/MerckBiosciences.

The standard ELISA test format applied was as follows:

96-well plates from Nunc or Corning were used which typically havecomparable quality with respect to lot-to-lot consistency. Forprecoating the plates with ganglioside antigens generally concentrationsof 500 ng/ml or 1000 ng/ml have been optimized to improve assay quality.Routinely, the test-specific antibodies are subject to biotinylationusing biotinylation kits from Amersham according to the standardoperation procedures recommended by the supplier. Streptavidinperoxidase conjugate is used for OD read-out. Generally, this procedureresults in highly sensitive and consistent test formats whereas whenusing the Generic Assays anti-ganglioside immuno-dot-blot testsdiffering results were observed, as pointed out above (see also FIG. 4and FIG. 5). This test format is designed for testing using biotinylatedIgM molecules. Therefore native, i.e. non-biotinylated IgMs were testedin a slightly modified ELISA test format using anti-kappa-chain antibodyalkaline phosphatase conjugate for OD read-out in order to be able tocompare native IgMs with biotinylated IgMs. Both ELISA test formats haveat first been compared with respect to specificity and sensitivity ofbinding to ganglioside antigen GD3, since positive reaction of thepresent recombinant IgM with GD3 was already established.

Both ELISA test formats confirmed positive binding of the IgM antibodyto GD3 in accordance with the Generic Assays anti-gangliosideimmuno-dot-blot (data not shown herein). There is apparently nointerference of biotinylation with respect to binding specifity. Asexpected, the ELISA test format using biotinylated IgM and streptavidinperoxidase OD read-out was slightly more sensitive than the one usingthe native IgM anti-kappa chain alkaline phosphatase OD read-out. Asthere was no indication that the biotinylation would interfere withbinding specificity the screening for binding specificities of therecombinant IgM antibody was continued using the standard ELISA testformat with biotinylated IgM and additional purified gangliosideepitopes.

The test results achieved with GM3 (data not shown herein) confirm thatthere is a clear positive binding specificity of the present IgMantibody for GM3, in accordance with the test results observed with theGeneric Assays blots. The sensitivity of binding to GM3 is comparable tothe sensitivity of binding to GD3 and is in the range of lower than 7ng/ml.

Surprisingly, further screening with respect to binding to purifiedganglioside antigens purchased from Calbiochem/Merck Biosciences led tounexpected results, namely to unveiling a positive binding of therecombinant IgM pentamer to GM1 and GD2 ganglioside antigens. In orderto confirm or exclude the first screening observations differentbiotinylated IgM concentrations were applied in the ELISA test format.The results are shown below in Table 1 (GM1) and Table 2 (GD2).

Clear positive, consistent and dilutable binding reactions to theganglioside antigens GM1 and GD2 were obtained which are in a comparableorder of sensitivity as compared to GD3 and GM3 binding, respectively. Aminor cross-reactivity with GD2 was previously visible from FIG. 5 too,but a cross-reactivity with GM1 was not visible at all from the resultsrepresented by FIG. 5.

The recombinant “1G6” CHO cell line producing the present IgM/GD3antibodies was deposited at the Health Protection Agency CultureCollection, Microbiology Services Division, Porton Down, UK, under HPACulture Collections Reference Number: Q9165 and Accession Number11062901. Hereinafter, it will be referred to as CHO cell line“IgM/Q9165”.

TABLE 1 Binding to GM1 ganglioside at different concentrations ofbiotinylated IgM BIOLISE Protocol description Name: NONAME.PRT MODIFIEDReader: SPECTRA Wavelength: 492 7 620 nm Lag time: 0 s Mode: NormalShaking: No Assay Description Data Name: D:\NTFSDATA\11494A.PLA ReadingType: Reader Reading Date: Nov. 22, 2011- Report Date: Oct. 29, 2012-13:48:17 15:13:37 Prompt #1: Precoating GM1 Prompt #4: Oct. 10, 2012[1000 ng/ml] Prompt #2: biotinilated IgM Prompt #5: Prompt #3: differentinternal Prompt #6: lots Comments: blanks row 1, 5 and 9; differentconcentrations of biotinilated IgM[H2,3,4,2000 ng/ml; [H 6,7,8,200ng/ml; and [H 10,11,12,20 ng/ml, 1:2 dilutions from H to A and readoutwith streptavidin POD conjugate. Lot from ganglioside, D00091608, fromCalbiochem Delta OD 1 2 3 4 5 6 7 8 9 10 11 12 A 0.002 0.103 0.074 0.0610.023 0.088 0.044 0.051 0.020 0.025 0.024 0.042 B 0.002 0.115 0.1090.085 0.021 0.052 0.055 0.057 0.018 0.025 0.029 0.074 C 0.002 0.1770.163 0.129 0.021 0.077 0.081 0.081 0.018 0.031 0.034 0.059 D 0.0030.268 0.288 0.235 0.023 0.123 0.138 0.140 0.019 0.046 0.042 0.056 E0.003 0.427 0.462 0.390 0.021 0.209 0.213 0.208 0.028 0.064 0.069 0.138F 0.010 0.827 0.802 0.650 0.032 0.361 0.366 0.358 0.021 0.117 0.1210.133 G 0.012 1.238 1.300 1.057 0.034 0.608 0.626 0.605 0.024 0.1740.183 0.194 H 0.016 1.905 1.985 1.678 0.037 1.022 1.031 1.016 0.0280.299 0.304 0.333

TABLE 2 Binding to GD2 ganglioside at different concentrations ofbiotinilated IgM BIOLISE Protocol description Name: NONAME.PRT MODIFIEDReader: SPECTRA Wavelength: 492 7 620 nm Lag time: 0 s Mode: NormalShaking: No Assay Description Data Name: D: \NTFSDATA\11228B.PLA ReadingType: Reader Reading Date: Report Date: Oct. 11, 2012- 13:08:49 Prompt#1: precoating GD2 Prompt #4: Oct. 10, 2012 gangliosid Prompt #2: wir1000n ng/ml, Prompt #5: biotinilated Prompt #3: IgM internal lot, Prompt#6: 020511-A Comments: blanks row 1, 5 and 9; rows 2-4 IgM biotinilated,lot: 20511-A [2000 ng/ml and rows 6-8 IgM, biotinilated, lot: 260511-A[200 ng/ml and rows 10-12 igM biotinilated, lot:80611-A with 20 ng/mland 1:2 dilutions H to A; readout with streptavidin POD conjugate DeltaOD 1 2 3 4 5 6 7 8 9 10 11 12 A 0.072 0.106 0.084 0.072 0.026 0.0470.148 0.015 0.022 0.025 0.029 0.044 B 0.080 0.138 0.126 0.098 0.0280.060 0.073 0.059 0.021 0.027 0.032 0.041 C 0.095 0.195 0.198 0.1560.036 0.102 0.093 0.089 0.022 0.034 0.036 0.055 D 0.070 0.298 0.3220.270 0.030 0.153 0.151 0.149 0.025 0.047 0.047 0.066 E 0.074 0.4920.520 0.408 0.031 0.235 0.240 0.234 0.023 0.072 0.068 0.089 F 0.1030.766 0.831 0.716 0.028 0.392 0.397 0.438 0.023 0.112 0.112 0.135 G0.092 1.305 1.361 1.144 0.031 0.672 0.695 0.655 0.023 0.183 0.184 0.197H 0.105 1.990 2.131 1.823 0.033 1.096 1.095 1.101 0.026 0.315 0.3130.330

Example 3

Basically any therapeutic or diagnostic potential of an antibody isdetermined by its potency to differentiate between desired and undesiredbinding targets, e.g. in the best case to exhibit reliable binding to abroad spectrum of malignant cells and no binding to normal phenotypes atall. Accordingly, it was decided to select some primary (normal) cellsand some well characterized malignant cell lines isolated from differenttumor tissues in order to investigate the binding patterns of presentIgM/GD3 antibody as expressed by CHO cell line IgM/Q9165. Since we didnot detect any differences in ganglioside binding patterns between thenative IgM/GD3 antibody and its biotinylated derivative it was decidedto use the biotinylated antibody for immunoimaging microscopy. Thedifferent cells investigated and the results of immunoimaging aresummarized in Table 3.

TABLE 3 Immunoimaging microscopy of human cells after contacting withbiotinylated IgM/GD3. Results of binding Cell Name Cell Type Originstudy MeWo; ATCC no. Adherent human skin/ positive HTB-65 fibroblastmalignant melanoma NCI-H69; ATCC multicell human lung/small cellpositive no. HTB-119 aggregates lung cancer, SCLC MDA-MB-231; adherenthuman metastatic positive ATCC no. HTB-26 epithelial breast cancer HTB14adherent human glioblastoma positive fepithelial ERL1690 adherent humanglioblastoma positive fibroblast huVEC adherent human umbilical veinnegative endothelial endothelium HDF5 IAM* adherent human lung negativefibroblastoid primary normal RPTEC IAM* adherent Human kidney immortal.negative epithelial normal *IAM—Institute of Applied Microbiology,Vienna Institute of Biotechnology

All cells were seeded into 8-well-chamber slides (μ-well ibi Treat,Microscopy Chambers) in sub-cultivation ratios and growth mediaaccording to the recommendations of the particular cell culturecollections from which they were purchased. According to the recommendedprotocols the cells should be still growing after an incubation time of1 to 2 days and cell density should not be too high in order to allowfor suitable immuno imaging. Most of the cell lines depicted in Table 3were growing strongly adherent which facilitated washing and fixationsteps without cell losses, except for both SCLC-cell lines (cor L88 andHTB 119) which only weakly adhered resulting in more sophisticatedwashing and fixation steps and some cell losses.

After incubation for a maximum of 2 days cell fixation was done asfollows:

-   -   a) cells have been treated twice with washing buffer (phosphate        buffered saline (PBS) containing 10% fetal calf serum);    -   b) after the washing step the cells have been covered with 3% of        paraformaldehyde solution in PBS for 15 minutes at room        temperature, for fixation;    -   c) after fixation the paraformaldehyde solution has been removed        and cells have been washed twice again. Wells have then been        covered with 10% FCS in PBS plus 0.3 molar glycine for 30        minutes at room temperature in order to saturate or block        unspecific binding reactions (e.g. free aldehyde groups        originating from the fixation with paraformaldehyde);    -   d) thereafter the blocking solution has been discarded and the        fixed cell layer has been incubated in parallel with either        biotinylated IgM/GD3 antibody (working concentration 10 ng/ml)        as positive control or with 10% FCS in OBS as negative control        for 1 hour at room temperature;    -   e) thereafter the incubation solutions have been decanted, the        wells rinsed twice with washing solution followed by incubation        with Qdot 525 streptavidin conjugate (working concentration 20        ng/ml) for approx. 1 hour;    -   f) in most experiments counter-staining of the cell nucleus with        DAPI has been applied. In such procedures the cells have again        been washed twice and thereafter incubated with DAPI solution        (200 nl DAPI dissolved in 10 ml PBS) for 15 minutes;    -   g) for microscopic immuno imaging the wells have been rinsed        again with PBS and then covered with PBS for analysis.

Binding of Qdot streptavidin to biotinylated IgM/GD3 resulted in greenlight emission signalling positive binding, which green light emissionwas automatically measured and corrected against unspecific backgroundby the software of the microscope. Staining or counter staining withDAPI lead to blue light emission. Generally, microscopic magnificationwas 600 times.

From the results of immuno imaging it was concluded that IgM/GD3antibody has indeed the potential to discriminate in binding betweenmalignant and non-malignant, i.e. “normal”, human eukaryotic cells.

Example 4: Investigation of Effector Functions Triggered by IgM/GD3Antibody

According to text book teachings of modern immune biology (see e.g.JANEWAY's IMMUNO BIOLOGY, 5^(th) edition, Kenneth Marghy, GarlandScience) IgM antibodies are expected to induce effector functions suchas apoptosis and activation of the complement cascade leading to killingof the respective target cells to which they bind. It is still unclearwhether they are also able to activate ADCCs (antibody dependendcytotoxic cells) as there are contradictory expert opinions which aredifficult to verify in standard animal models. At present, reliableresults can only be expected and obtained from clinical trials withhuman volunteers afflicted with cancer.

Nevertheless, in order to test the potency of the IgM/GD3 pentamericantibody to kill certain target cells via activation of the humancomplement cascade a complex in vitro test system simulating an evenmore complex in vivo situation was established comprising the followingprocedural steps:

a) Culturing of target cells was done in a cell culture medium asrecommended by the respective cell culture collection from which thecell lines have been obtained and supplemented with 20% of heatinactivated normal human serum (hiNHS). The heat inactivation (56° C.for 1 hour) destroys or inactivates at least some of the proteinsinvolved in the activation of the complement cascade. Supplementationwith hiNHS was thus used as a reference standard marker for 100% cellpropagation in a human serum environment.b) Supplementation of cell culture medium with 20% of normal human serum(NHS), i.e. without heat-inactivation, in replacement of hiNHS, as asource of human complement proteins (of which approximately 30 differentproteins have been identified in the prior art). NHS was thus intendedto represent the composition of human blood from which all cellularcomponents have been removed while all soluble constituents have beenleft essentially unchanged.c) Addition of the IgM/GD3 antibody at different concentrations to thetarget cell culture supplemented with NHS and determination of thepercentage of surviving cells as compared to the cell culturessupplemented with hiNHS or NHS, respectively, but without addition ofthe IgM/GD3 antibody. Antibody concentrations tested varied in a rangeof from 1 ug/ml to 100 ug/ml. Specific antibody concentrations selectedcomprised 1, 2, 4, 10, 20, 40 and 100 ug/ml (see FIGS. 6-8)

In FIGS. 6 through 8 the results of an in vitro test series with threedifferent target cell lines are shown (for identification of targetcells see Table 3 above). It can be taken therefrom that supplementationof the cell culture medium by 20% of NHS as a source of complement waseffective in causing the lysis of some target cells depending on theconcentration of the anti-GD3 antibody IgM/GD3 added to the respectivecell cultures.

The fact that replacement of hiNHS by NHS in the absence of antibodyalso exhibits some cell lysis or growth inhibition is not surprising andis a well-known though not yet well understood phenomenon.

Anyway, from the results achieved by immunoimaging of “malignant” and“normal” cells summarized in Table 3 above as well as from the resultsof in vitro testing of complement activation it is concluded that thepresent antibody IgM/GD3 is well suited for both diagnostic andtherapeutic use, e.g. for diagnostic formulations to detect cancer cellsin biopsies or blood samples, as well as for therapeutic applications totreat various cancers. The present invention therefore also relates tothe use of the present anti-GD3 IgM antibody as a diagnostic tool in thecourse of in vitro or in vivo cancer diagnostics of human individuals.The invention further relates to the use of the present anti-GD3 IgMantibody as an active ingredient in a pharmaceutical composition ormedicament for therapeutical application in the treatment of cancer.

While the present invention has been exemplified with human recombinant“1G6” IgM/GD3 antibody obtainable by CHO dhfr⁻ cell line IgM/Q9165deposited at the Health Protection Agency Culture Collection,Microbiology Services Division, Porton Down, UK under HPA CultureCollections Reference Number: Q9165 and Accession Number 11062901, it isunderstood that the present invention also encompasses other anti-GD3IgM antibodies that have essentially the same binding characteristics ashereinbefore disclosed of deposited antibody IgM/GD3 and/or that competewith said antibody IgM/GD3 for binding to specific epitopes and whichother antibodies have been obtained via other routes.

For example, the present invention also relates to IgM/GD3-likeantibodies that have been genetically or chemically modified,particularly at the light and heavy chain variable regions, by usingstate of the art techniques known to the skilled artisan such asmodification, deletion, insertion or substitution of genetic informationleading to modified nucleotide and/or amino acid sequences as comparedto present sequences SEQ ID NO: 1 and SEQ ID NO: 2, to the extent thatthe resulting IgM antibodies typically share at least 90%, preferably atleast 95% sequence homology of their respective heavy and/or light chainvariable regions with either or both of the amino acid sequences SEQ IDNO: 1 and SEQ ID NO: 2, and in addition share essentially the samespecific binding characteristics with the presently disclosed andclaimed CHO antibody IgM/GD3. The present invention also relates to theuse of the present IgM/G3 antibody as a screening tool in thedevelopment of anti-anti-idiotype antibodies. More specifically, in oneembodiment the invention relates to anti-anti-idiotype antibodiesobtained by using present antibody IgM/GD3 or fragments thereof asscreening tools in a first procedural step in a screening setup for thedetection of antibodies that compete with IgM/GD3 for specific bindingto one or more of the ganglioside epitopes mentioned hereinbefore.Preferably, such anti-anti-idiotype IgM antibodies in their heavy and/orlight chain variable regions share at least 90%, and more preferably atleast 95% of sequence homology with either or both of the correspondingidiotype peptide sequences SEQ ID NO: 1 and SEQ ID NO: 2.

What is claimed is:
 1. A CHO cell line producing a recombinant humanmonoclonal IgM antibody, which comprises in its heavy chain variableregion a polypeptide amino acid sequence SEQ ID NO:1, and in its lightchain variable region a polypeptide amino acid sequence SEQ ID NO:2, theIgM further comprising the following characteristics: capability ofoligospecific binding to purified ganglioside epitopes GD3, GM3, GD2 andGM1; and capability of specific binding to malignant cancer cellsselected from the group consisting of melanoma cells, small cell lungcancer cells, glioblastoma cells, estrogen receptor-negative metastaticbreast cancer cells, said CHO cell line being deposited under accessionnumber 11062901 at the Health Protection Agency Culture Collection,Microbiology Services Division, Porton Down, UK.